1TYM Sage Grouse
Samples arrived Samples checked in against list .
Extractions
Dilute and Array fullITS Primers
Primers arrived 100uM in 50 ul
Add 575 to get primers to 8 uM with Integra Pipette using Pipette/Mix setting
Add 87.5 ul TE to new Primer plates
Use Nimbus to array first twelve plates using FULLITS_Primer_Prep_Adjustable
TRNL PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 450 | 1350 |
0.45 | 10M dNTPs | 450 | 202.5 |
0.3 | Kapa HiFi HotStart DNA Pol | 450 | 135 |
7.25 | HPLC H2O | 450 | 3263 |
11 | Total Volume | 450 | 4950 |
Add 11 ul to each well of a hard shell, full skirt plate. Add 2 uL of primers and 2uL of template to each well.
fullITS PCR MasterMix
ul/rxn | Reagent | # of rxns | ul needed |
|
|---|---|---|---|---|
3 | 5X Kapa HiFi Buffer | 450 | 1350 | 600 |
0.45 | 10M dNTPs | 450 | 202.5 | 90 |
0.3 | Kapa HiFi HotStart DNA Pol | 450 | 135 | 60 |
8.25 | HPLC H2O | 450 | 3713 | 1650 |
12 | Total Volume | 450 | 5400 | 2400 |
Plate | TRNL Primers | fullITS Primers |
|---|---|---|
2TRNL1 | TRNL19 | fullITS05 |
TRNL20 | fullITS06 | |
2TRNL2 | TRNL17 | fullITS07 |
TRNL18 | fullITS08 | |
2TRNL3 | TRNL21 | fullITS09 |
TRNL22 | fullITS10 | |
2TRNL4 | TRNL23 | fullITS11 |
TRNL24 | fullITS12 | |
2TRNL5 | TRNL16 | fullITS02 |
Template Format:
Run TRNL plates on thermocycler program TRNL_T*:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Denature | 95 | 35X | 0:30 |
Annealing | 55 | 35X | 0:30 |
Extension/Elongation | 72 | 35X | 0:30 |
Hold | 4 | 1X | 0:00 |
Run fullITS plates on thermocycler program fullITS:
Step | Temp C | Cycles | Time |
|---|---|---|---|
Denature | 95 | 1X | 10:00 |
Denature | 94 | 35X | 0:30 |
Annealing** (Row C) | 55 | 35X | 0:40 |
Extension/Elongation | 72 | 35X | 1:00 |
Final Extension | 72 | 1X | 10:00 |
Hold | 4 | 1X | 0:00 |
MagBead Cleanup:
Equilibrate Beads to room Temperature
Add 15uL of ultra pure water to each well.
Add 24 ul of MagBeads to each well.
Pipette mix up and down 10 times.
Incubate at RT for 5 minutes
Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)
Remove 65 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well
Reaspirate from each well to assure maximum EtOH removal
Allow plate to air dry for 7 minutes.
Remove sample plate from magnet plate.
Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.
Place sample plate back on magnet for 5 minutes or until all wells are cleared.
Transfer 40 ul to labeled transparent plate (Plate name_PCR_MIDs)
Normalize to 5 ng/ul
qPCR
Sequencing
Pool 2 parts TRNL with 2 parts fullITS and 1 part Baldwin CO1-Lark.
TRNL starting 2.77
FI starting 1.62
BALD starting 3.23
1 nM 1TYM_TRNL: 36 ul pool with 64 ul RSB
1 nM 1TYM_FI: 62 ul pool with 38 ul RSB
1 nM 1BALD_CO1: 31 ul pool with 69 ul RSB
Load Pool:
18 ul 1nM 1Bald
36 ul 1nM 1TYM_FI
36 ul 1nM 1TYM_TRNL
9 ul 1 nM PhiX
1 ul RSB